The aim of this proposal is to determine if specific DNA base alterations induced by environmental carcinogens can cause critical mutations in a target gene leading to its activation and subsequent initiation of neoplastic process. The objectives are: (i) to understand the role of mutagenic DNA base modifications specifically incorporated within a defined region of ras proto- oncogene without subjecting the target cells to direct carcinogen exposure; (ii) identify the sequence changes of the modified region responsible for altered gene function and cellular phenotype; and (iii) compare the response of NIH/3T3 and human cells in in vitro transformation by transfection with specifically altered cloned genes. These studies will provide an insight on the role of specific adduct and site related critical mutational activation of normal cellular genes in the process of neoplastic cellular transformation and lead to the development of a more relevant assay system using human cells as the recepeints of transfected oncogenes. The approach to be employed in this study is as follows. The cloned ras gene (plasmid pbc-N1) will be site specifically modified to solely contain principal mutagenic lesions of DNA alkylation (O6-ethylguanine or O4-ethylthymine) targeted within a specific coding regions of the gene by AMV DNA polymerase. The effect of each of the lesions will be studied in the induction of mutations at critical sites of ras gene and their relative potency in conferring the transforming properties upon this gene. Mutated plasmid will be introduced into the mouse NIH/3T3 and human fibroblast cells by calcium phosphate mediated DNA transfection and co-transfection with other genes. Neoplastic phenotype conferred by establishment of modified gene in the transformants will be determined by scoring the morphologically altered foci, anchorage-independent cellular growth and tumorigenicity in nude mice.